Aminopterin dosage forms and methods for inflammatory disorders

ABSTRACT

Embodiments of the present invention provide dosage forms and methods for treating a patient with an inflammatory disorder with a therapeutically effective amount of aminopterin, or a pharmaceutically acceptable salt thereof, that achieve efficacy without concomitant toxicity. Within certain embodiments, the present invention provides a method for treating an inflammatory disorder in a patient with uninterrupted doses of aminopterin.

BACKGROUND OF THE INVENTION

Aminopterin, or 4-amino-pteroyl-L-glutamic acid, is a potent antifolate[see Franklin, U.S. Pat. No. 2,575,168]. Synthesized in 1946 by theLederle Laboratories, a division of American Cyanamid Co., aminopterinwas marketed in 0.5 mg tablets in 1953 for the treatment of childhoodleukemia. In 1965, the marketing of aminopterin as a pharmaceutical inthe United States ceased.

In 1951, Gubner treated patients with rheumatoid arthritis, psoriaticarthritis, uncomplicated psoriasis, and atopic dermatitis with 0.75-2mg/day for durations of approximately 1 week and greater, or cumulativeweekly doses of greater than 5.25 mg [Gubner et al., Am. J. Med. Sci.22:176, 1951; and Gubner, Arch. Derm., Chicago 64:688, 1951]. Althoughsome patients improved, the majority of patients developed toxicreactions including stomatitis, nausea, diarrhea, and alopecia thatnecessitated discontinuation of the drug. Gubner concluded that “thetoxic effects of aminopterin place practical limitations on its use as atherapeutic agent” [Gubner et al., Am. J. Med. Sci. 22:176, 1951].

In 1955, Rees et al. treated 171 patients with psoriasis with fivedosage schedules using the 0.5 mg tablet that comprised: (i) 1 tabletdaily for six days; (ii) 1 tablet daily for six days, one week rest,then 1 tablet daily for six days; (iii) 1 tablet daily for 12 days; (iv)2 tablets daily for six days; and (v) 2 tablets daily for three days,then 1 tablet daily for six days [Rees et al., AMA Arch. Derm.72(2):133-43, August 1955]. A rapid clearing of psoriatic lesions werenoted, with toxic reactions occurring with a frequency of 0%, 2.5%, 13%,and 30% on schedules (i), (ii), (iii) and (iv), respectively. Too fewpatients were treated with schedule (v) to assess. Toxic reactionsincluded stomatitis, alopecia, and leucopenia. When the above scheduleswere concluded, the psoriatic lesions invariably recurred, usuallywithin weeks. Some patients were given multiple courses of the aboveschedules, with rest periods between courses.

In 1958, Edmundson and Guy treated patients with a schedule comprising 1tablet daily for six days, withdrawal for three days, and again dailyfor six days, for a total of 6 mg in 12 doses [Edmundson and Guy, AMAArch. Derm. 78(2):200-3, August 1958]. Improvement was noted, andremissions typically lasted several months.

In 1959, Rees and Bennett report the treatment of 329 patients withpsoriasis using the same schedules from their 1955 study [Rees andBennett, J. Invest. Dermatol. 32(1):61-66, January 1959). Courses of theschedules were repeated in some patients every 3 weeks to once every 3years, although it is not disclosed which schedules were repeated. Theoverall incidence of toxicity was 21% and the most common toxicreactions were stomatitis and intensification of the lesions, followedby alopecia, GI disturbances, and leucopenia. The authors noted thattreatment should be discontinued at the slightest hint of a toxicreaction.

In 1961, Rees and Bennett compared the effect of daily doses of 0.5 mgaminopterin against daily doses of 2.5 mg methotrexate using the sameschedules from their 1955 study [Rees and Bennett, Arch. Dermatol.83:970-72, June 1961]. In 1963, Strakosh also compared the effect ofdaily doses of 0.5 mg aminopterin against daily doses of 2.5 mgmethotrexate according to schedules comprising: (i) 1 tablet daily for12 days, followed by one-week's rest, and repeated as often as deemedadvisable; (ii) 1 tablet daily for 3 days followed by three-days' restperiod and repeated until 12 tablets in all were given, and thenfollowed by one-week's rest period and repeated as often as deemedadvisable; and (iii) any variation of (i) and (ii) above [Strakosch,Dermatologica 126:259-267, 1963]. Both concluded that methotrexate isless toxic and less efficacious than aminopterin in treating psoriasis.However, the doses compared were not equipotent to one another, withmethotrexate being used in an amount 4-fold less than would be requiredto be equipotent with aminopterin. Thus, by modern standards noconclusions could be drawn regarding the actual relative efficacy,toxicity, or therapeutic index of these antifolates. Later, Rees et al.suggest the opposite, that methotrexate may be more safe and efficaciousthan aminopterin in treating psoriasis [Rees et al., Arch. Dermatol.90:544-52, December 1964].

In 1964, Rees et al. review the literature on the standard of practicewith aminopterin in treating psoriasis, describing all known schedulesof administration [Rees et al., Arch. Dermatol. 90:544-52, December1964]. In all cases, dosing schedules were similar, being comprised ofdaily dosings of tablets for periods greater than 1 week until efficacyor toxicity was observed, at which time dosing was interrupted by restperiods of varying duration.

The prior art are also contains several reports of using aminopterin toreduce inflammation in animal models. In 1952, Gubner et al.demonstrated the efficacy of cumulative weekly doses of 0.3 mg/kgaminopterin in the rat formaldehyde arthritis model [Gubner et al., J.Invest. Dermatol. 19(4):297-305, October 1952]. In 1964, Pagedemonstrated the efficacy of cumulative weekly doses of 0.35 mg/kgaminopterin in the rabbit dermal inflammation model, but in the processthe animals became severely leucopenic and one subject died [Page, Ann.N.Y. Acad. Sci. 116:950-63, Aug. 27, 1964]. In 1986, Galivan et al.demonstrated the efficacy of cumulative weekly doses of 0.12 mg/kgaminopterin in the rat adjuvant arthritis model, but the animalssuffered from severe toxicity [Galivan, et al., Methotrexate in adjuvantarthritis, in Chemistry and Biology of Pteridines 1986. Pteridines andFolic Acid Derivatives, B. A. Cooper and V. M. Whitehead, Editors. 1986,Walter de Gruyter & Co.: Berlin. p. 847-49]. Similarly, in 2000Andersson et al. demonstrated the efficacy of cumulative weekly dosesof >1.5 mg/kg aminopterin in the rat antigen-induced arthritis model,but again the animals suffered from severe toxicity [Andersson, et al.,Eur. J. Pharm. Sci. 9(4):333-43, 2000].

SUMMARY OF THE INVENTION

It has been discovered in the instant invention described herein dosagesand methods for treating a patient with an inflammatory disorder with atherapeutically effective amount of aminopterin, or a pharmaceuticallyacceptable salt thereof, that provide unexpected advantages of patientconvenience without creating concomitant toxicity manifestations. Inparticular, it has been discovered that aminopterin can be used inuninterrupted cycles of doses as an anti-inflammatory agent, and in someembodiments at doses lower than previously taught in the art.

One aspect of the invention is a method for treating inflammatoryconditions with aminopterin without creating unacceptable toxicitymanifestations. Another related embodiment is a method of treatingbronchopulmonary dysplasia, canine atopic dermatitis, and bovine acutepneumonic pasteurellosis with an antifolate, and aminopterin inparticular. Another related embodiment is a method of treatinginflammatory bowel disease with aminopterin.

Further, it has been discovered that aminopterin doses can beadministered in dosage forms containing less aminopterin per tablet thandescribed by earlier publications. These lower doses are achieved by thedevelopment of tablets containing less than 0.5 mg aminopterin, whichallow lower doses to be administered to patients and also allow theability to tailor these lower doses more accurately to a particularpatient's body weight.

Another aspect of the invention is treatment of rheumatoid arthritis,psoriatic arthritis and atopic dermatitis in humans with a weekly doseof aminopterin of 5.25 mg or less. A related embodiment of the inventionis a method of dosing a human with psoriasis with a cumulative weeklydose of less than 2 mg/kg aminopterin. A related embodiment is a dosageform of aminopterin containing less than 0.5 mg per tablet. It will beappreciated by one in the art the above identified aspects of theinvention avoid the toxicity and irregular dosing schedules ofaminopterin that were a major inconvenience to patients withinflammatory disorders. Another aspect of the invention are tabletscontaining less than 0.5 mg aminopterin per tablet, which make itpossible to administer lower doses and also make it easier to tailorlower doses to a particular patient's weight in order to avoid toxicity.

Further still, antifolates and aminopterin in particular have beendiscovered to be efficacious in the treatment of bronchopulmonarydysplasia, and inflammatory disorders that occur naturally in domesticand agricultural animals, and in some embodiments with only a singledose of aminopterin. In particular, embodiments of the present inventionprovide:

A method for treating an inflammatory disorder in a patient, comprisingadministering to said patient a therapeutically effective amount ofaminopterin, or a pharmaceutically acceptable salt thereof, inuninterrupted cycles. In preferred embodiments, the number ofuninterrupted cycles is at least 24. In yet other preferred embodiments,the periodicity of the uniterrupted cycles is weekly, wherein the numberof doses in each cycle is 2, and more preferably 1. In some embodiments,a second drug is used in combination therapy. Folic acid is particularlypreferred as the second drug.

Typically, each cycle consists of less than 0.07 mg aminopterin perkilogram of patient body weight that can be administered in less than 5tablets. Optimal dosing without toxicity manifestations is achieved inparticularly preferred embodiments utilizing one or more tabletscontaining less than 0.5 mg aminopterin each.

In other embodiments, a method is provided for treating an inflammatorydisorder in a patient, comprising administering to said patient a singledose of a therapeutically effective amount of an antifolate, or apharmaceutically acceptable salt thereof. In preferred embodiments, theantifolate is aminopterin. Preferred inflammatory disorders that can betreated with a single dose of an antifolate include humanbronchopulmonary dysplasia, canine atopic dermatitis and bovine acutepneumonic pasteurellosis. Canine atopic dermatitis is one of the mostcommon conditions seen by veterinarians, and bovine acute pneumonicpasteurellosis, also known as ‘shipping fever’ in transported cattlecosts the cattle industry $1 billion per year (Malazdrewich, et al. Vet.Pathol. 38:297, 2001). In shipping fever, the ability to treat affectedanimals with the minimum number of drug administrations has criticaleconomic advantages.

In other embodiments, a method is provided for treating an inflammatorydisorder in a patient, comprising administering to said patient atherapeutically effective amount of aminopterin, or a pharmaceuticallyacceptable salt thereof, wherein the therapeutically effective amount ofaminopterin administered per week is less than 0.07 mg aminopterin perkilogram of patient body weight. In this method, aminopterin ispreferably administered in one or more tablets, wherein at least onetablet contains less than 0.5 mg aminopterin.

In other embodiments, a method is provided for treating an inflammatorydisorder in a patient, comprising administering to said patient atherapeutically effective amount of aminopterin, or a pharmaceuticallyacceptable salt thereof, wherein the inflammatory disorder is treatedmore rapidly than with methotrexate. The rate of treatment is quantifiedwith a disease manifestation scoring system selected from the groupconsisting of ACR20, ACR50, ACR70, ACR-N, JRA30% DOI, JRA50% DOI, JRA70%DOI, PASI, plumonary function testing, oxygen saturation, lesionalscores, and pruritis scores. In other embodiments, the rate of treatmentis quantified by the area under the efficacy-time curve.

In other embodiments, a pharmaceutical composition is providedcomprising a therapeutically effective amount of aminopterin, or apharmaceutically acceptable salt thereof, wherein the pharmaceuticalcomposition contains less than 0.5 mg aminopterin. The pharmaceuticalcomposition is preferably a tablet, and in certain embodiments is a 0.1mg or 0.2 mg tablet. In a particularly preferred embodiment, the activepharmaceutical ingredient is substantially free of impurities.

These and other aspects of the present invention will become apparentupon reference to the detailed description and illustrative exampleswhich are intended to exemplify non-limiting embodiments of theinvention. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a dose-response plot of aminopterin and methotrexate in themurine air-pouch model of inflammation.

FIG. 2 is a plot showing the efficacy of aminopterin and methotrexatedays after the last dose of each drug in the murine air-pouch model ofhuman inflammation.

FIG. 3 is a plot showing the relative efficacy of aminopterin andmethotrexate in the rat oxygen-toxicity model of human bronchopulmonarydysplasia.

GLOSSARY

Prior to setting forth the invention in detail, it may be helpful to anunderstanding thereof to set forth definitions of certain terms thatwill be used hereinafter.

The term “active pharmaceutical ingredient” as used herein means amixture of an antifolate and impurities resulting from one or moreorganic synthetic steps. An organic synthetic step will comprise thepartial or complete transformation of one or more chemicals to one ormore new chemicals, and will also usually entail at least one or morepurification steps to enrich the one or more new chemicals. The one ormore purification steps will consist of methods known to those skilledin the art, such as, for example, crystallization, extraction, andchromatography. Ideally, purification steps are not required, or the oneor more purification steps will enrich a single new chemicalpreferentially. For example, an active pharmaceutical ingredientcontaining aminopterin is made by the transformation of2,4-diamino-6-(bromomethyl)pteridine and N-(4-aminobenzoyl)-L-glutamicacid in dimethylacetamide to aminopterin plus several impurities,wherein the impurities comprise at least folic acid and untransformed2,4-diamino-6-(bromomethyl)pteridine and N-(4-aminobenzoyl)-L-glutamicacid.

The term “aminopterin purity” as used herein means the percentage ofantifolate in an active pharmaceutical ingredient or pharmaceuticalcomposition, wherein the antifolate is aminopterin (see glossarydefinition of “impurities”).

The term “antifolate” as used herein means a molecule and/or metabolitesof the molecule that interfere with the normal metabolism or utilizationof folic acid (i.e. folate) and/or metabolites of folic acid in acell-free biochemical system or in cells found in cell culture, tissueculture, leukemia, cancer, a mammal, and a human. For example,aminopterin and methotrexate, as well as their polyglutamatedmetabolites, are antifolates. Typically, an antifolate and/or metaboliteof the antifolate will interfere with the normal metabolism orutilization of folic acid and/or metabolites of folic acid by blockingtheir binding to one or more enzymes or receptors that include, forexample, the reduced folate receptor, the folic acid receptor,folylpolyglutamate synthase, dihyrofolate reductase, thymidylatesynthase, methylene-tetrahydrofolate reductase, amidophosphoribosyltransferase, glycinamide ribonucleotide transformylase,aminoimidazole carboxamide ribonucleotide transformylase, andhomocysteine methyltransferase. Examples of folic acid metabolites thatan antifolate and/or metabolites of the antifolate will interfere withinclude, but are not limited to, 5-methyl-tetrahydrofolate-(glu)_(n),5,10-methylene-tetrahydrofolate-(glu)_(n), tetrahydrofolate-(glu)_(n),N-5-fomamino-tetrahydrofolate-(glu)_(n),5,10-methenyl-tetrahydrofolate-(glu)_(n),10-formyl-tetrahydrofolate-(glu)_(n), and5-formyl-tetrahydrofolate-(glu)_(n), where -(glu)_(n) refers to theglutamates attached to the metabolite and n is the number of attachedglutamates. When n=1, no glutamates have been added to these folic acidmetabolites beyond that found in the original folic acid molecule. Whenn is greater than 1 these folic acid metabolites are considered to bepolyglutamates (see glossary term below).

The term “AUC” as used herein means the area under the plasmaconcentration-time curve for a single dose of a drug as described morefully in Shargel and Yu, Applied Biopharmaceutics and Pharmacokinetics,4^(th) Edition, 1999, Appleton & Lange, Stamford, Conn., incorporatedherein by reference. The AUC is proportional to the amount of drug thatreaches the plasma.

The term “combination therapy” as used herein refers to the use of twoor more therapeutics according to a therapeutic protocol with the aim ofproviding a highly optimized treatment plan to most effectively treat ainflammatory disorder in a patient.

The term “disease manifestation” as used herein refers to any undesiredresult of a inflammatory disorder. Particular disease manifestationsinclude, but are not limited to lethargy, joint pain, jointinflammation, joint damage, inflammatory cells in joint fluid, andpsoriatic skin lesions. Disease manifestations are often quantified inthe art using well known scoring systems, such as for example, ACR20,ACR50, ACR70 or ACR-N in rheumatoid arthritis (Felson et al., ArthritisRheum. 38:727, 1995; Bathon et al., N. Engl. J. Med. 343:1586, 2000; andWilliam St. Clair et al., Arthritis Rheum. 50(11):3432-3443, 2004);JRA30% DOI, JRA50% DOI and JRA70% DOI in juvenile rheumatoid arthritis(Giannini et al., Arthritis Rheum. 40(7): 1202-1209, 1997; and Lovell etal., Arthritis Rheum. 48(1):218, 2003), the PASI (Psoriasis Activity andSeverity Index) score in psoriasis (Finlay et al., Br. J. Dermatol.123:751, 1990); pulmonary function testing and oxygen saturation inbronchopulmonary dysplasia; and lesional and pruritis scores in canineatopic dermatitis (Olivry and Mueller, Veterinary Dermatol. 14:121-146,2004). Other ways of quantifying disease manifestations in a patientwith an inflammatory disorder will be familiar to those skilled in theart.

The term “efficacy” as used herein means an antifolate istherapeutically effective. Generally, a greater level of efficacy willbe achieved by increasing the dose and/or frequency of administration ofan antifolate given to a population, such that a greater proportion ofthe population will receive a benefit and/or there will be a greatermagnitude of benefit in an individual patient. If a first antifolate ismore potent than a second antifolate, it will reach a greater level ofefficacy than the second antifolate using identical amounts of each.

The term “impurities” as used herein refers to the impurities found inthe active pharmaceutical ingredient. Impurities arise during theorganic synthetic steps employed in the preparation of the activepharmaceutical ingredient, and in the case of aminopterin include, forexample, folic acid, pterins, and conjugates of p-aminobenzoic acid(i.e. pABAglu). Impurities may be the result of incompletetransformation of chemicals during an organic synthetic step, or one ormore side-reactions that result in chemicals being transformed intounintended new chemicals. As defined herein, the pharmaceuticallyacceptable carriers and optional therapeutic ingredients in apharmaceutical composition do not constitute impurities. Impurities in apharmaceutical composition pertain only to those impurities in theactive pharmaceutical ingredient used to make the pharmaceuticalcomposition. Impurities are quantitated using any measurable propertysuitable for quantitating molecules. Such measurable properties will befamiliar to those in the art and include, for example, HPLC peak area(i.e. “area”), mass and moles. Impurities will typically be convenientlyquantitated based on their area, but may also be quantitated accordingto their weight or moles using, for example, uv absorbance of collectedHPLC peak fractions and the known extinction coefficient and molecularweight of each impurity. If the molecular weight of an impurity isunknown, mass spectrometry may be used. The percentage of an impurity inan active pharmaceutical ingredient or pharmaceutical composition is theamount of the impurity divided by the total amount of impurities plusantifolate multiplied by 100, wherein all impurities and the antifolateare quantitated using the same measurable property. For example, thepercentage of an impurity in an active pharmaceutical ingredient orpharmaceutical composition may be expressed as an area %, weight %, ormole %. In a specific example, if an impurity constitutes 0.1 micromoleof an active pharmaceutical ingredient and the aminopterin plus totalimpurities in the active pharmaceutical ingredient together constitute 1micromole, the percentage of the impurity in the active pharmaceuticalingredient is 10 mole %. In another example, if an impurity is 0.25 areaunits of a pharmaceutical composition and the total impurities plusantifolate together are 1.0 area units of the pharmaceuticalcomposition, the percentage of the impurity in the pharmaceuticalcomposition (or the active pharmaceutical ingredient) is 25 area %. In afurther example, if an impurity is 0.04 mg of a pharmaceuticalcomposition containing 2 mg of the active pharmaceutical ingredient, thepercentage of the impurity in the pharmaceutical composition is 2 weight%. The percentage of total impurities may be obtained by summing thepercentages of all individual impurities in the active pharmaceuticalingredient or pharmaceutical composition, wherein all the individualimpurities are quantitated using the same measurable property. Thepercentage of antifolate in an active pharmaceutical ingredient orpharmaceutical composition is the amount of antifolate divided by thetotal amount of impurities plus antifolate multiplied by 100, whereinall impurities and the antifolate are quantitated using the samemeasurable property. Thus, the percentage of total impurities in theactive pharmaceutical ingredient or pharmaceutical composition mayalternatively be obtained by subtracting the percentage of antifolatefrom 100%.

The term “oral bioavailability” as used herein refers to the fraction ofan antifolate dose given orally that is absorbed into the plasma after asingle administration to a patient. A preferred method for determiningthe oral bioavailability is by dividing the AUC of an antifolate dosegiven orally by the AUC of the same antifolate dose given intravenouslyto the same patient, and expressing the ratio as a percent. Othermethods for calculating oral bioavailability will be familiar to thoseskilled in the art, and are described in greater detail in Shargel andYu, Applied Biopharmaceutics and Pharmacokinetics, 4^(th) Edition, 1999,Appleton & Lange, Stamford, Conn., incorporated herein by reference.

The term “patient” is an animal or a human.

The term “pharmaceutical composition” as used herein means the activepharmaceutical ingredient combined with one or more pharmaceuticallyacceptable carriers, and optionally other therapeutic ingredients.Suitable pharmaceutically acceptable carriers will be familiar to thoseskilled in the art, and will comprise, for example, microcrystallinecellulose, lactose, silicon dioxide, croscarmellose, sodium benzoate,sorbitol, magnesium stearate and flavoring. The active pharmaceuticalingredient will typically comprise only a small percentage of the totalpharmaceutical composition. For example, a “2 mg aminopterin tablet” isa pharmaceutical composition that weighs about 100 mg, and comprisesabout 98 grams of pharmaceutically acceptable carriers and about 2 mg ofthe active pharmaceutical ingredient. The 2 mg of the activepharmaceutical ingredient consists mostly of aminopterin and a smallfraction of impurities (see glossary definition of “impurities” and“active pharmaceutical ingredient”). If this pharmaceutical compositionis said to be “substantially free of impurities”, then the smallfraction of impurities will be, for example, less than 5 area %, lessthan 5 weight %, or less than 5 mole % percent of the activepharmaceutical ingredient (see glossary definition of “substantiallyfree of impurities”).

The term “polyglutamates” as used herein refers to folate and antifolatemetabolites that have attached two or more glutamates. The enzymefolylpolyglutamate synthase attaches additional glutamates to folatemetabolites and some antifolates beyond the glutamate that is on theoriginal folate metabolite and some antifolates to form a polyglutamatechain. Examples of aminopterin and methotrexate polyglutamates includeaminopterin-(glu)_(n) and methotrexate-(glu)_(n), where -(glu)_(n)refers to the glutamates attached to the antifolate and n is the numberof attached glutamates. When n=1, no glutamates have been added beyondthat in the original antifolate molecule. When n is greater than 1 theseantifolate are considered to be polyglutamates, and thus have apolyglutamate chain. A polyglutamate chain is said to have a length,wherein a first polyglutamate chain is said to have a longer length thana second polyglutamate chain if the n of the first polyglutamate chainis larger than the n of the second polyglutamate chain. Folate andantifolate metabolites having polyglutamate chains are often refered toas polyglutamated species or polyglutamates. A mixture of polyglutamatechains having different lengths is said to comprise polyglutamate chainlengths. Both aminopterin and methotrexate are metabolized topolyglutamates having an n of from 2 to about 5, as described in greaterdetail in, Gangjee et al., Curr. Med. Chem. Anti-Canc. Agents. 2(3):331,2002, incorporated herein by reference.

The term “potency” as used herein means the effectiveness of a dosage toachieve a particular level of efficacy. The dosage takes into accountthe amount of drug given at each administration (i.e. dose), thefrequency of administration, and optionally, the total number of dosesto be given. The effectiveness of a dosage can be quantitated bymeasuring the cumulative amount of drug administered in a defined period(i.e. the “dose rate”) of the dosage and that results in a particularlevel of efficacy, where effectiveness and potency are inversely relatedto the dose rate. A first drug whose dosage has greater effectiveness orpotency than the dosage of a second drug is said to be more potent thanthe second drug. For example, a first antifolate is more potent than asecond antifolate if both achieve the same level of efficacy using adose rate that is smaller in the first antifolate than the secondantifolate. In a more specific example, a first antifolate is morepotent than a second antifolate if both achieve the same level ofefficacy using a dosage that is identical except for the dose of thefirst antifolate being smaller than the dose of the second antifolate.In a further example, a first antifolate is more potent than a secondantifolate if both achieve the same level of efficacy using a dosagethat is identical except for the frequency of administration of thefirst antifolate being smaller than the frequency of administration ofthe second antifolate. Two different antifolates may have differentpotencies, and will therefore achieve the same level of efficacy atdifferent therapeutically effective amounts. For example, aminopterin isabout 25 times more potent than methotrexate, and therefore thetherapeutically effective amount of aminopterin required for a level ofefficacy is about 25-fold less than the therapeutically effective amountof methotrexate required for the same level of efficacy. Thetherapeutically effective amounts of two antifolates (i.e. either as adosage or as a single dose) that result generally in the same level ofefficacy are referred to herein as equi-potent. For example, two oraldoses of 2 mg/m² aminopterin are approximately equi-potent to four oraldoses of 25 mg/m² methotrexate.

The term “substantially free of impurities” as used herein means thatthe percentage of total impurities in an active pharmaceuticalingredient or in a pharmaceutical composition is less than five percent(see definition of “impurities”). For example, a pharmaceuticalcomposition or active pharmaceutical ingredient is said to besubstantially free of impurities if the percentage of total impuritiesis less than 5 area %, 5 weight % or 5 mole %. As used herein,“substantially free of impurities” also means that the percentage ofantifolate in an active pharmaceutical ingredient or in a pharmaceuticalcomposition is ninety-five percent or greater (see definition of“impurities”). For example, a pharmaceutical composition that has apercentage of antifolate with an area %, weight %, or mole % equal to orgreater than ninety-five, is said to be substantially free ofimpurities. In a more specific example, a pharmaceutical composition oractive pharmaceutical ingredient that has an aminopterin purity with anarea %, weight %, or mole % equal to or greater than ninety-five, issaid to be substantially free of impurities.

The term “therapeutic component” as used herein refers to atherapeutically effective dosage (i.e. dose and frequency ofadministration) that eliminates, reduces, or prevents the progression ofa particular disease manifestation in a percentage of a population. Anexample of a commonly used therapeutic component is the ED₅₀, whichdescribes the dose in a particular dosage that is therapeuticallyeffective for a particular disease manifestation in 50% of a population.

The term “therapeutic index” as used herein refers to the ratio of aparticular toxicity component (i.e. the dose that is toxic in apercentage of a population, for example, the TD₅₀) to a particulartherapeutic component (i.e. the dose that is effective in percentage ofa population, for example, the ED₅₀). It will be understood by a skilledpractitioner that the determination of the therapeutic index of anantifolate, such as aminopterin, need not be determined for everypatient in a population with a inflammatory disorder treated accordingto the invention. It is sufficient to use a representative number ofpatients with the inflammatory disorder to establish a therapeutic indexfor the entire patient population with the inflammatory disorder, whereat least 5 patients with the inflammatory disorder will be required inorder to establish a therapeutic index for the entire patient populationwith the inflammatory disorder.

The term “therapeutic protocol” as used herein refers to a schedule ofdosing, routes of administration, and schedule duration for two or moredrugs that are employed within combination therapy. The schedule may befurther divided into specific phases each of a specified duration. Forexample, the phases of modern rheumatoid arthritis treatment involvingthe treating with NSAIDs and various other small-molecule or biologicDMARDS either sequentially or together constitute specific phases of atherapeutic protocol. During each phase, the type of drugs to be given,their dosing, and routes of dosing are defined for all patients ingeneral or may be modified based on other disease factors such alaboratory or imaging tests.

The term “therapeutically effective amount” as used herein means thedosage (dose or amount, and frequency) of antifolate which directly orindirectly kills inflammatory cells, arrests the accumulation ofinflammatory cells, or reduces the accumulation of inflammatory cells ina human or other mammal afflicted with an inflammatory inflammatorydisorder such as, for example, arthritis of undefined etiology,rheumatoid arthritis, juvenile rheumatoid arthritis, atopic dermatitis,bronchopulmonary dysplasia, inflammatory bowel disease, psoriaticarthritis and psoriasis, or a animal with, for example, canine atopicdermatitis or bovine acute pneumonic pasteurellosis. The term as usedherein shall also mean the dosage of an antifolate which directly orindirectly reduces or increases the activity of molecules secreted byinflammatory and/or non-inflammatory cells participating in aninflammatory inflammatory disorder in a human or mammal, such that theamount of antifolate arrests, reduces, or eliminates altogether thedegree of pathologic inflammation associated with the inflammatoryinflammatory disorder. Typically, a therapeutically effective amountwill also eliminate, reduce, or prevent the progression of, one or moredisease manifestations. A skilled artisan readily recognizes that inmany cases antifolates may not provide a cure, but may only providepartial benefit. Furthermore, the skilled artisan recognizes thatbecause individual patients and disease states may vary, some patientsmay receive little, or no benefit at all. A dosage of antifolate that“kills”, “arrests”, “reduces” or “eliminates” as described above, in aleast some patients, is considered therapeutically effective. The dosemagnitude of a therapeutically effective amount of aminopterin in theacute or chronic management of a inflammatory disorder will vary withthe severity of the inflammatory disorder to be treated and the route ofadministration. The dosage and dose rate of aminopterin will depend on avariety of factors, such as the weight and calculated surface area ofthe patient, the specific pharmaceutical composition used, the object ofthe treatment, i.e., therapy or prophylaxis, the nature of the diseaseto be treated, the judgment of the treating physician, and the responseof the individual patient. In general, a therapeutically effectiveamount of aminopterin will be a dose of aminopterin from 0.001-0.07mg/kg, 0.005-0.03 mg/kg, and most preferably 0.010-0.03 mg/kg, given asa single or divided dose. Patients may be upward titrated from below towithin these dose ranges to a satisfactory control of diseasemanifestations. Once improvement in the patient's condition hasoccurred, a maintenance dosage of a composition of this invention isadministered, if necessary. Subsequently, the dose rate may be reducedby reducing the dose or frequency of administration, or a combination ofboth, as a function of the symptoms, to a level at which the improvedcondition is retained. When the symptoms have been alleviated to thedesired level, the practitioner may elect to cease treatment. Patientsmay, however, require intermittent treatment upon any recurrence ofdisease symptoms, or prophylactically scheduled treatments as required.The therapeutically effective amount of aminopterin may optionally beadministered prior to, contemporaneous with, or after at least onetherapeutically effective dose of leucovorin or folic acid.

The term “toxicity component” as used herein refers to the dosage (i.e.dose and frequency of administration) required to produce a toxicitymanifestation in a percentage of a population. An example of a commonlyused toxicity component is the TD₅₀, which describes the dose in aparticular dosage required to produce a toxicity manifestation in 50% ofa population. Generally, there is an inverse relationship between doseand frequency necessary to produce the same toxicity component, whereless antifolate given more often will produce the same toxicitycomponent as more antifolate given less often.

The term “toxicity manifestation” as used herein refers to any undesiredeffect of a drug. A drug is said to be toxic or have toxicity if itcauses a toxicity manifestation in a percentage of a population. Forantifolates, particular toxicity manifestations include, but are notlimited to mucositis, alopecia, diarrhea, myelosuppression,nephrotoxicity, hepatotoxicity, severe hepatotoxicity, neurotoxicity,and death. Toxicity manifestations also include indirect indications oftoxicity such as thrombocytopenia (e.g. <50,000/μL), neutropenia (e.g.<750/μL), elevated liver enzymes (e.g. >5 times normal), erythrocyteantifolate (e.g. eAMT and eMTX), the necessary discontinuation oftherapy by a patient because of the inability to endure furthertreatment with the drug, or increased hospital admissions due to theinability to continue therapy. Thus, a practitioner can assess thepresence and the magnitude of a particular toxicity manifestation. Many,if not all, of the toxicity manifestations of antifolates can bereversed by the prior, contemporaneous or subsequent administration ofleucovorin, a reduced folate that is well known in the art to rescueantifolate toxicity.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention provide dosages and methods fortreating a patient with an inflammatory disorder with a therapeuticallyeffective amount of aminopterin, or a pharmaceutically acceptable saltthereof, that provide unexpected advantages of patient convenience whilealso achieving efficacy without concomitant toxicity. The inflammatorydisorder may occur in humans and comprise, for example, arthritis ofundefined etiology, rheumatoid arthritis, psoriatic arthritis, juvenilerheumatoid arthritis, psoriasis, inflammatory bowel disease, atopicdermatitis, bronchopulmonary dysplasia. The animal inflammatory disordermay occur in animals and comprise, for example, arthritis, canine atopicdermatitis and bovine acute pneumonic pasteurellosis.

Within certain embodiments, the present invention provides a method fortreating an inflammatory disorder in a patient with uninterrupted cyclesof aminopterin doses, wherein the doses are a therapeutically effectiveamount. Uninterrupted means that aminopterin doses are repetitivelyadministered to a patient for at least 4 cycles, 12 cycles, 24 cycles,and most preferably greater than 52 cycles, wherein the periodicity ofthe cycles is constant, and wherein the greatest duration between thelast dose of one cycle and the first dose of the next cycle does notexceed 21 days, 14 days, and most preferably 7 days. Within thisdefinition, ‘periodicity of the cycles is constant’ means that theduration between corresponding doses in consecutive cycles is constantto within a 12 hour range. For example, if the periodicity is denoted tobe 7 days (i.e. 168 hours), then according to the present invention thephrase ‘periodicity of the cycles is constant’ will be construed to meanthat the duration between corresponding doses in consecutive cycles mayrange from 162 to 174 hours. Further within this definition, the numberof aminopterin doses in each cycle can range from 1 to 5, and eachindividual dose may comprise taking one or a plurality of individualdosage forms.

Thus, in a preferred embodiment 1 dose of aminopterin is administered toa patient every 7 days for at least 4 cycles, and most preferably for atleast 52 cycles (i.e. a year). In this case, the number of doses percycle is only a single dose, the periodicity is 7 days, and the greatestduration between the last dose of one cycle and the first dose of thenext cycle is 6 days. In another preferred embodiment one dose ofaminopterin is administered on Monday and one on Tuesday for at least 52cycles. In this case, the number of doses per cycle is 2, theperiodicity is 7 days, and the greatest duration between the last doseof one cycle and the first dose of the next cycle is 5 days (i.e.Wednesday through Sunday). In yet another preferred embodiment, a doseof aminopterin is administered in the morning and another at night on aparticular day of the week by taking two tablets with each dose, thiscycle is then repeated for at least 52 cycles. In this case, the numberof doses per cycle is 2 where each dose comprises taking 2 dosage forms,the periodicity is 7 days, and the greatest duration between the lastdose of one cycle and the first dose of the next cycle is 6 days (i.e.the days between the day of the week the doses is given). It will beunderstood that other schedules are within the embodiments of theinvention. For example, in one embodiment, one dose of aminopterin isadministered on Monday and one on Wednesday for at least 52 cycles. Inthis case, the number of doses per cycle is 2, the periodicity is 7days, and the greatest duration between the last dose of one cycle andthe first dose of the next cycle is 4 days (i.e. Thursday throughSunday). In the most preferred embodiment, the periodicity is weekly(i.e. 7 days).

In other embodiments, methods are provided for treating an inflammatorydisorder in a patient comprising administering a single cycle ofaminopterin in a therapeutically effective amount. Most preferably,bronchopulmonary dysplasia in a human and acute pneumonic pasteurellosisin a cow is effectively treated with a single cycle of aminopterin,wherein the number of doses in the cycle is 1.

In other embodiments, methods are provided for treating an inflammatorydisorder in a patient more rapidly than it can be treated withmethotrexate by administering to the patient one or more doses ofaminopterin in a therapeutically effective amount. In particular, atherapeutically effective amount of aminopterin will eliminate, reduce,or prevent the progression of, one or more disease manifestations in thepatient more rapidly than a therapeutically effective amount ofmethotrexate. While the rate to reach a particular efficacy can beachieved more rapidly with aminopterin than with methotrexate in someembodiments, it will be understood that the final maximum level ofefficacy achieved may be the same or different. In preferredembodiments, the final level of efficacy achieved will be greater foraminopterin than for methotrexate, in addition to this level beingreached more rapidly by aminopterin. The rate can be measured using anyquantitative endpoint of disease activity (see ‘disease manifestation’and ‘therapeutically effective amount’ in the Glossary). In preferredembodiments, the rate is quantified as a function of time by measuringany of the following scoring system: ACR20, ACR50, ACR70, ACR-N, JRA30%DOI, JRA50% DOI, JRA70% DOI, PASI, pulmonary function testing, oxygensaturation, lesional scores, and pruritis scores. In other preferredembodiments, the rate is quantified by documenting an increase in thearea under the ‘efficacy-time curve’, wherein the curve is establishedby plotting the output parameter of the scoring system as a function oftime {see for example, Bathon et al., N. Engl. J. Med. 343:1586, 2000for an example of this approach). The area under the efficacy-time curveis not to be confused with the ‘AUC’ defined in the Glossary for thearea under the plasma concentration-time curve for a single dose of adrug.

In still further embodiments the weekly dosage comprises a cumulativedose of aminopterin ranging from 0.001-0.07 mg/kg, 0.005-0.03 mg/kg, andmost preferably 0.010-0.03 mg/kg. For a typical 60 kg adult, the weeklydosage thus comprises a cumulative dose of aminopterin ranging from0.06-4 mg, 0.3-1.8 mg, and most preferably 0.6-1.8 mg. In otherpreferred embodiments where the dosage form is a 0.1 mg, 0.2 mg, 0.5 mgor 1.0 mg tablet, the weekly dosage comprises taking one to threetablets by mouth of any combination thereof. The above cumulative weeklydose of aminopterin can be given either in a single administration at aparticular time, or as a plurality of administrations during a singleday, or over multiple days. Using the methods of the instant invention,it has been discovered that aminopterin can be given to a patient withan inflammatory disorder without toxicity manifestations, and in themost preferred embodiments without interruption.

Embodiments of the present invention further provide methods fortreating an inflammatory disorder in a patient using combinationtherapy, which comprises administering to said patient a therapeuticallyeffective amount of aminopterin, or a pharmaceutically acceptable saltthereof, according to a therapeutic protocol involving at least oneother therapeutic. The at least one other therapeutic may beadministered prior to, contemporaneous with, or after administering theaminopterin. The at least one other therapeutic also includes a singledosage form containing aminopterin and at least one other therapeutic, amultiple dosage form, wherein the aminopterin and the at least one othertherapeutic are administered separately, but concurrently, or a multipledosage form wherein the two components are administered separately, butsequentially. The at least one other therapeutic can be, for example,folic acid, leucovorin, dextromethorphan, memantine, prednisone, a cox-2inhibitor, a non-steroidal anti-inflammatory drug, vincristine,dexamethasone, asparaginase, daunorubicin, mercaptopurine, etoposide,cytarabine, doxorubicin, cisplatin, ifosfamide, paclitaxel,5-fluoruracil, dianydrogalacitol, tamoxifen, piperazinedione,mitoxantrone, diaziquone, aminothiadiazole, methotrexate, tenoposide,vincristine, echinomycin, 6-mercatopurine, dexamethasone,cyclophosphamide, soluble TNF receptors, antibodies, and humanizedantibodies.

In preferred embodiments, a dose of aminopterin from 0.001-0.066 mg/kg,0.005-0.03 mg/kg, and most preferably 0.010-0.03 mg/kg, is suitable foruse in a therapeutic protocol employed during a combination therapy. Insome embodiments, aminopterin can be directly substituted formethotrexate in a therapeutic protocol employing methotrexate byadministering aminopterin at about 4-8% of the dose of methotrexate inthe protocol. This substitution yields at least the same level ofefficacy and therapeutic index as methotrexate, but with far fewertablets taken by the patient.

In one embodiment, aminopterin is substituted for methotrexate in thetreatment of adult rheumatoid arthritis in a therapeutic protocolemploying another non-steroidal anti-inflammatory drug by administeringa single weekly oral dose of 0.5 to 2 mg aminopterin instead of a singleweekly dose of 7-25 mg methotrexate. In another embodiment, aminopterinis substituted for methotrexate in the treatment of juvenile rheumatoidarthritis in a therapeutic protocol employing another non-steroidalanti-inflammatory drug by administering a single weekly oral dose of 0.5to 2 mg/m² aminopterin instead of a single weekly dose of 7-25 mg/m²methotrexate. In still another embodiment, psoriasis in an adult istreated in a therapeutic protocol by administering a single weekly oraldose of 1 to 4 mg aminopterin instead of a single weekly dose of 15-25mg methotrexate, or as two weekly oral doses of 1 to 2 mg aminopterininstead of two weekly doses of 7-13 mg methotrexate.

An embodiment of the present invention further provides methods oftreating an inflammatory disorder in a human, which comprisesadministering to said human an active pharmaceutical ingredientsubstantially free of impurities, wherein the antifolate in the activepharmaceutical ingredient is a therapeutically effective amount ofaminopterin, or a pharmaceutically acceptable salt thereof. In someembodiments, the impurities may include, for example, folic acid,pterins, and conjugates of p-aminobenzoic acid. In preferredembodiments, the aminopterin purity in the active pharmaceuticalingredient is equal to or greater than 95 area %, equal to or greaterthan 95 weight %, or equal to or greater than 95 mole % (see“aminopterin purity” in glossary). In other embodiments, the activepharmaceutical ingredient has less than 5 area %, less than 5 weight %,or less than 5 mole % impurities, and more preferably less than 3 area%, less than 3 weight %, or less than 3 mole % impurities. In preferredembodiments, administering an active pharmaceutical ingredientcontaining an aminopterin dose from 0.03 mg/m² to about 2 mg/m², or0.001-0.066 mg/kg will provide a therapeutically effective amount ofaminopterin and will be substantially free of impurities.

The chemical synthesis of an active pharmaceutical ingredientsubstantially free of impurities and containing aminopterin can beperformed by several different sequences of organic synthetic steps. Itis understood that one or ordinary skill in the art would be able tomake an active pharmaceutical ingredient substantially free ofimpurities and containing aminopterin in light of the followingdisclosure, including the Examples, and information known to those ofordinary skill in the chemical synthesis field. For example, beginningwith readily available starting materials, an active pharmaceuticalingredient substantially free of impurities and containing aminopterinmay be synthesized according to Scheme 1.

As illustrated above, the commercially available2,4,5,6-tetraminopyrimidine, compound 1, may be condensed withβ-bromopyruvaldoxime to provide 2,4-diamino-6-(bromomethyl)pteridine,compound 2 [see Taghavi-Moghadam and Pfleiderer, Tet. Lett. 38:6835,1997 and Taylor and Portnoy, J. Org. Chem. 38:806, 1973]. Alternatively,compound 1 may be reacted with 1,3-dihydroxyacetone to provide2,4-diamino-6-pteridinemethanol, compound 5 [see Baugh and Shaw, J. Org.Chem. 29:3610, 1964]. Compound 5 is purified and reacted with HBr anddibromotriphenylphosphorane (Ph₃PBr₂) in dimethylacetamide to affordcompound 2 [see Piper and Montgomery, J. Org. Chem. 42:208, 1977; Piperand Montgomery, J. Heterocycl. Chem. 11:279, 1974; Piper and Montgomery,U.S. Pat. No. 4,077,957; and Piper and Montgomery, U.S. Pat. No.4,079,056]. In still other embodiments, compound 2 can be arrived at viathe reaction of compound 1 with 1,1-dichloroacetone to form2,4-diamino-6-(methyl)pteridine, which is then reacted with bromide [seeCatalucci, U.S. Pat. No. 4,224,446].

Regardless of the route to its synthesis, compound 2 is condensed withcommercially available N-(p-aminobenzoyl)-L-glutamic acid, compound 3,in dimethylacetamide to afford the active pharmaceutical ingredientsubstantially free of impurities and containing aminopterin, compound 4,as the antifolate [see Piper and Montgomery, J. Org. Chem. 42:208, 1977;Piper and Montgomery, U.S. Pat. No. 4,077,957; Piper and Montgomery,U.S. Pat. No. 4,079,056; and Catalucci, U.S. Pat. No. 4,224,446].

In other embodiments, an active pharmaceutical ingredient substantiallyfree of impurities and containing aminopterin as the antifolate may beobtained by purification of aminopterin preparations contaminated withimpurities by, for example, ion-exchange chromatography or by HPLC [seeHeinrich et al., J. Am. Chem. Soc. 75:5425, 1953 and Tong et al., Lancet2:719, 1975]. In still other embodiments, an active pharmaceuticalingredient substantially free of impurities and containing aminopterinas the antifolate may be obtained by the direct transformation (i.e.amination) of folic acid to aminopterin and subsequent purification byHPLC [see Zimmermann, U.S. Pat. No. 4,767,859].

An embodiment of the present invention further provides pharmaceuticalcompositions substantially free of impurities and comprising an activepharmaceutical ingredient, wherein the antifolate in the activepharmaceutical ingredient is a therapeutically effective amount ofaminopterin, or a pharmaceutically acceptable salt thereof. In someembodiments, the impurities may include, for example, folic acid,pterins, and conjugates of p-aminobenzoic acid. In preferredembodiments, the pharmaceutical composition has less than 5 area %, lessthan 5 weight %, or less than 5 mole % impurities, and more preferablyless than 3 area %, less than 3 weight %, or less than 3 mole %impurities. In other embodiments, the aminopterin purity in thepharmaceutical composition is equal to or greater than 95 area %, equalto or greater than 95 weight %, or equal to or greater than 95 mole %.In preferred embodiments, a pharmaceutical composition containing from0.2 mg to about 2.0 mg aminopterin will provide a therapeuticallyeffective amount of aminopterin and will be substantially free ofimpurities. In particularly preferred embodiments, a pharmaceuticalcomposition contains 0.5 mg aminopterin will provide a therapeuticallyeffective amount of aminopterin and will be substantially free ofimpurities.

The aminopterin purity of the active pharmaceutical ingredient is usedto established how much active pharmaceutical ingredient is required inthe pharmaceutical composition to obtained a desired final dose ofaminopterin, or a pharmaceutically acceptable salt thereof, in thepharmaceutical composition. For example, if a pharmaceutical compositionis to contain about 1 mg of aminopterin and the aminopterin purity inthe active pharmaceutical ingredient is 95 weight %, then about 1.0526mg of the active pharmaceutical ingredient will be required in thepharmaceutical composition to provide about 1 mg of aminopterin.

In addition to the active pharmaceutical ingredient, pharmaceuticalcompositions substantially free of impurities contain one or morepharmaceutically acceptable carriers (see glossary definition of“pharmaceutical composition”). Pharmaceutical compositions substantiallyfree of impurities are most readily prepared by combining an activepharmaceutical ingredient substantially free of impurities, whereinaminopterin is the antifolate in the active pharmaceutical ingredient,in intimate admixture with one or more pharmaceutical carriers accordingto conventional pharmaceutical compounding techniques.

The carrier may take a wide variety of forms depending on the form ofthe pharmaceutical composition (i.e. “preparation” or “form”) desiredfor administration, e.g., oral or parenteral (including intravenousinjections or infusions). In preparing the pharmaceutical composition inan oral dosage form any of the usual pharmaceutical carriers may beemployed. Usual pharmaceutical carriers include, for example, water,glycols, oils, alcohols, flavoring agents, preservatives, coloringagents, and the like in the case of oral liquid preparations (such asfor example, suspensions, solutions, and elixirs); aerosols; or carrierssuch as starches, sugars (e.g. lactose), microcrystalline cellulose,diluents, granulating agents, lubricants, binders, disintegrating agentsand the like, in the case of oral solid preparations (such as forexample, powders, capsules, and tablets) with the oral solidpreparations generally being preferred over the oral liquidpreparations. For pediatric patients, however, it will be appreciated tothose skilled in the art that pleasant tasting oral liquid preparationsare preferred.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage form in adults, in which case solidpharmaceutical carriers are employed. If desired, tablets may be coatedby standard aqueous or nonaqueous techniques. The parenteral dosage formcan consist of a sterile solution of the active ingredient, either inits free or salt form, in physiological buffer or sterile water. Inaddition, parenteral solutions can contain preservatives such asbenzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, a standard reference text in this field. Inembodiments employing parenteral, oral liquid, or other aqueouscompositions, care must be taken since electrophilic substitution bywater converts aminopterin to folic acid, and such preparations havebeen noted to accumulate folic acid to undesirable levels over thecourse of six months of storage. Accordingly, such aqueous compositionsare best stored desiccated and hydrated within several hours to severaldays prior to patient administration.

In addition to the common dosage forms set out above, the pharmaceuticalcompositions of the present invention may also be administered bycontrolled release means and/or delivery devices such as those describedin U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 3,630,200;4,008,719; 4,687,660 and 4,769,207, the disclosures of which are herebyincorporated by reference.

Optionally, the pharmaceutical composition contains other therapeuticingredients. Such therapeutic ingredients may be added to amelioratecertain side-effects, particularly those of aminopterin, or add topatient convenience by reducing the number of dosage forms that must betaken. Suitable therapeutic ingredients for combining with thepharmaceutical composition may include, for example, folic acid,leucovorin, dextromethorphan, memantine, prednisone, a cox-2 inhibitor,a non-steroidal anti-inflammatory drug, vincristine, dexamethasone,asparaginase, daunorubicin, mercaptopurine, etoposide, cytarabine,doxorubicin, cisplatin, ifosfamide, paclitaxel, 5-fluoruracil,dianydrogalacitol, tamoxifen, piperazinedione, mitoxantrone, diaziquone,aminothiadiazole, methotrexate, tenoposide, vincristine, echinomycin,6-mercatopurine, dexamethasone, cyclophosphamide, soluble TNF receptors,antibodies, and humanized antibodies.

As used in the methods and compositions of the present disclosure, theterm “pharmaceutically acceptable salts” refers to salts prepared frompharmaceutically acceptable non-toxic acids or bases including inorganicacids and bases and organic acids and bases. The sodium or di-sodiumsalts of aminopterin are pharmaceutically acceptable salts ofaminopterin.

Since aminopterin is both basic and acidic, salts may be prepared frompharmaceutically acceptable non-toxic acids or bases including inorganicand organic acids or inorganic and organic bases. Such salts may containany of the following anions: acetate, benzensulfonate, benzoate,camphorsulfonate, citrate, fumarate, gluconate, hydrobromide,hydrochloride, lactate, maleate, mandelate, mucate, nitrate, pamoate,phosphate, succinate, sulfate, tartrate and the like. Such salts mayalso contain the following cations: aluminum, calcium, lithium,magnesium, potassium, sodium, zinc, benzathine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine, and procaine.

Unless the route of administration is specified in a method disclosedherein, any suitable route of administration may be employed forproviding a human with a therapeutically effective amount ofaminopterin, or a pharmaceutically acceptable salt thereof. For example,oral, rectal, parenteral, transdermal, subcutaneous, intramuscular, andthe like may be employed as appropriate. Dosage forms include tablets,coated tablets, troches, dispersions, suspensions, solutions, caplets,capsules, patches, and, the like. Pharmaceutical compositions includethose suitable for oral, rectal and parenteral (including subcutaneous,intramuscular, and intravenous) administration, although the mostsuitable route in any given case will depend on the nature and severityof the inflammatory disorder being treated. The most preferred route ofthe present invention is the oral route. The pharmaceutical compositionsmay be conveniently presented in unit dosage form, and prepared by anyof the methods well known in the art of pharmacy.

Pharmaceutical compositions of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets, or tablets, or aerosols sprays, each containing a predeterminedamount of the pharmaceutically active ingredient, as a powder orgranules, or as a solution or a suspension in an aqueous liquid, anon-aqueous liquid, an oil-in-water emulsion, or a water-in oil liquidemulsion. Such pharmaceutical compositions may be prepared by any of themethods of pharmacy, but all methods include the step of bringing intoassociation the active pharmaceutical ingredient with at least onepharmaceutical carrier. In general, the pharmaceutical compositions areprepared by uniformly and intimately admixing the active pharmaceuticalingredient with liquid pharmaceutical carriers or finely divided solidpharmaceutical carriers or both, and then, if necessary, shaping theproduct into the desired presentation.

For example, a tablet may be prepared by compression or molding,optionally, with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing in a suitable machine the activepharmaceutical ingredient in a free-flowing form such as powder orgranules, optionally mixed with a binder, lubricant, inert diluent,surface active or dispersing agent. Molded tablets may be made bymolding in a suitable machine, a mixture of the powdered compoundmoistened with an inert liquid diluent. Desirably, each tablet containsfrom about 0.5 mg to about 2 mg of aminopterin or a therapeuticallyacceptable salt thereof, and each cachet or capsule contains from about0.5 mg to about 2 mg of aminopterin or a therapeutically acceptable saltthereof. Most preferably, the tablet, cachet or capsule contains eitherone of two dosages, about 0.5 mg or about 1 mg of aminopterin or atherapeutically acceptable salt thereof.

In still other embodiments, pharmaceutical compositions contain atherapeutically effective amount of aminopterin, or a therapeuticallyacceptable salt thereof, with a standard deviation of less than 5%, andmore preferably a standard deviation of less than 3%. The standarddeviation is a measure of dose uniformity (i.e. consistency) in thepharmaceutical compositions with a smaller standard deviation being anindication of greater dose uniformity. The pharmaceutical compositionsmay be different portions of a single pharmaceutical composition from asingle formulation batch, or may be from multiple pharmaceuticalcompositions of the same dosage form that are each the result ofdifferent formulation batches. Thus, the standard deviation can be ameasure of dose uniformity both within the same batch and betweenbatches.

The standard deviation of an antifolate in pharmaceutical compositionsis determined by measuring the amount of antifolate in a known amount ofeach pharmaceutical composition using methods familiar to those in theart. For example, the amount of aminopterin and methotrexate in apharmaceutical composition can be quantitated using scanning uvspectrophotometry, HPLC, or a radioligand DHFR binding assay [see Kamenet al., Anal. Biochem. 70:54, 1976 and Ratliff et al., J. Clin. One.16:1458, 1998]. As defined herein, calculation of a standard deviationrequires measuring aminopterin from at least two randomly selected partsof a single batch of a pharmaceutical composition, or from each of atleast two different batches of a pharmaceutical composition.

The invention is further defined by reference to the following examplesdescribing in detail, the methods, and use and preparation of thepharmaceutical compositions of the present invention. It will beapparent to those skilled in the art, that many modifications, both tomaterials, and methods, may be practiced without departing from thepurpose and interest of this invention.

EXAMPLES

In Examples 1-15 that follow, “aminopterin” is the antifolate in anactive pharmaceutical ingredient employed in a pharmaceuticalcomposition substantially free of impurities.

Example 1 Efficacy in the Murine Air-Pouch Model of Inflammation

To identify the optimum dose of aminopterin for treating rheumatoidarthritis and to quantitate its potency relative to methotrexate, wetested aminopterin and methotrexate in a murine air-pouch model ofinflammation whose dose-response relationship to methotrexate has beenshown by Cronstein et al. to correlate exceedingly well with thedose-response relationship of methotrexate in humans [Cronstein, B. N.,D. Naime, and E. Ostad, The anti-inflammatory mechanism of methotrexate.Increased adenosine release at inflamed sites diminishes leukocyteaccumulation in an in vivo model of inflammation. J. Clin. Invest.,1993. 92(6):2675-82].

Accordingly, groups of 5 animals each (10-15 week-old male mice) weregiven a fixed dose (mg/kg) per group of aminopterin (0.0005, 0.001,0.002, 0.005, 0.008, 0.010, 0.050) or methotrexate (0.01, 0.02, 0.05,0.08, 0.10, 0.50, 1.00) by weekly ip injection on days 1, 7, 14 and 21.A separate group of 5 animals was given vehicle by ip injection on thesame days (0.9% saline, control, n=5). On day 16, air pouches wereinduced on the animals by injecting 3 ml of air subcutaneously on theback. On day 22 (i.e. one day after the last dose of drug or vehicle),inflammation was induced by injection of 1 ml of a suspension ofcarrageenan (2% weight/volume in calcium- and mangnesium-free PBS). Themice were put back in their cages and allowed to run free for 4 hours.The mice exhibited no signs of toxicity at the end of the dosing period.After 4 hours, the animals were sacrificed, the pouches flushed with 2ml of PBS and the exudates harvested. Aliquots were diluted 1:1 withmethylene blue (0.01% w/v in PBS), and the cells were counted.

These studies demonstrated that aminopterin and methotrexate diminishedin a dose-dependent fashion the number of neutrophils that accumulatedin the carrageenan-treated air pouches with EC₅₀s of 0.0009 mg/kg/wk and0.04 mg/kg/wk, respectively (FIG. 1). Thus, aminopterin was found to be43-fold more potent than methotrexate (P<0.001). That efficacy ofaminopterin can be obtained at such low weekly doses has not beenreported in the prior art. Weekly doses in the above range are known tobe many-fold below the maximally tolerated weekly dose of aminopterin,where mucositis just starts to appear [Ratliff, A. F., et al., Phase Iand pharmacokinetic trial of aminopterin in patients with refractorymalignancies. J. Clin. Oncol., 1998. 16(4): 1458-64].

Example 2 Duration of Efficacy in the Murine Air-pouch Model ofInflammation

Although the 43-fold difference in EC₅₀s between aminopterin andmethotrexate in their anti-inflammatory activity was felt to be mostlikely due to the preferential uptake efficiency of aminopterin, we alsoconsidered the possibility that a difference in the rate of drugexcretion from cells could be contributing to this difference byessentially increasing the magnitude of the “trough” of one drug overanother prior to each weekly dose. We therefore examined the rate atwhich anti-inflammatory activity was lost in the air-pouch model afterthe cessation of 4 weeks of dosing at the maximally efficacious dose(FIG. 2).

Accordingly, animals (10-15 week-old male mice) were given weekly ipinjections of vehicle (0.9% saline, control, n=60), aminopterin (n=60,0.05 mg/kg) or methotrexate (n=60, 1.0 mg/kg) over the course of a monthon days 1, 7, 14 and 21. On day 16, air pouches were induced on theanimals by injecting 3 ml of air subcutaneously on the back. On day 25the air pouches were re-inflated with 1.5 ml of air. Pouches werere-inflated as needed to maintain them. On days 22 (+1), 24 (+3), 26(+5), 29 (+8), 33 (+12), and 43 (+22) inflammation was induced in agroup of n=5 animals from the vehicle, aminopterin and methotrexate armsof the study by injection of 1 ml of a suspension of carrageenan as a 2%weight/volume solution in calcium-free and mangnesium-free PBS (numbersin parentheses indicate days after the fourth and last ip injection ofeither vehicle or drug). After 4 hours, the mice were sacrificed, thepouches flushed with 2 ml of PBS and the exudates harvested. Aliquotswere diluted 1:1 with methylene blue (0.01% w/v in PBS), and the cellswere counted.

The data showed no statistically signficant difference between the rateof recovery of inflammation at 8, 12 or 22 days after the lastantifolate dose, and revealed that the duration of action of bothaminopterin and methotrexate was quite long, consistent with the longintracellular half-lives of polyglutamated antifolates. This exampleshows that the greatest duration between the last dose of one cycle andthe first dose of the next cycle can be up to 21 days for aminopterin.

Example 3 Efficacy in the Rat Oxygen-toxicity Model of Inflammation

To identify the optimum dose of aminopterin for treatingbronchopulmonary dysplasia in humans and to quantitate its potencyrelative to methotrexate, we tested aminopterin and methotrexate in amurine oxygen-toxicity model of inflammation [Deng, H.; Mason, S. N.;Richard L. Auten, J. Lung inflammation in hyperoxia can be prevented byantichemokine treatment in newborn rats. American Journal of RespiratoryCritical Care Medicine 2000, 162, 2316-2323].

Briefly, timed pregnant rats were placed in air (two litters) or 95%oxygen (one litter) the day of delivery after recombining litters amongfour just delivered dams. Litter sizes were the same. Aminopterin (0.2mg/kg), methotrexate (0.35 mg/kg) or vehicle were then introduced by theip route on day 2 after birth. Dams were rotated daily between air andoxygen cages to avoid maternal oxygen toxicity. On day 8, the pups wereweighed and then euthanized, tracheas are cannulated and the pulmonaryartery is perfused with saline. Bronchoalveolar lavage fluid (BALF) wasthen obtained for cell count and chemokine analysis (FIG. 3). The lungswere removed and snap frozen for myeloperoxidase activity assay(neutrophil accumulation within lung parenchyma) and 8-isoprostane, amarker of lipid oxidation. The pulmonary artery was perfused and thelungs were inflation-fixed with buffered formalin at 25 cm H₂O.

As expected, the data showed that hyperoxia resulted in a marked influxof inflammatory cells, predominantly comprised of neutrophils (PMNs).The data showed a statistically significant reduction in the number ofneutrophils after only a single 0.2 mg/kg injection of aminopterin 6days prior to sacrificing the animals. In contrast, methotrexate at17-times the dose of aminopterin (0.35 mg/kg), failed to reduce theinflux of neutrophils. Efficacy of aminopterin can be obtained using asingle low dose. This example shows that bronchopulmonary dysplasia canbe treated with an antifolate, and that achieving efficacy occurs at amuch more rapid rate for aminopterin than for methotrexate. This dose ismany-fold lower than the maximally tolerated dose of aminopterin, whichproduces mucositis [Ratliff, A. F., et al., Phase I and pharmacokinetictrial of aminopterin in patients with refractory malignancies. J. Clin.Oncol., 1998. 16(4): 1458-64].

Example 4 2,4-Diamino-6-pteridinemethanol (5) hydrobromide salt

2,4,5,6-Tetraminopyrimidine.H₂SO₄.H₂O (75.0 g, 0.293 mole) was added toa stirred solution of BaCl₂.2H₂O (71.5 g, 0.293 mole) in H₂O (1.45 l.)at 85-90° C. The mixture was stirred rapidly at about 90° C. for 15 min,cooled to 40° C., and filtered from BaSO₄, which was washed thoroughlyon a funnel with H₂O. The clear, yellow filtrate was then dilutedfurther with H₂O to give a volume of 4.35 l. This solution of thetetraminopyrimidine.2HCl was then added to a solution of NaOAc (4.35 l.of 4 N) in which dihydroxyacetone (79.3 g, 0.88 mole) andcysteine.HCl.H₂O (51.5 g, 0.293 mole) had just been dissolved. Theresulting solution was stirred mechanically at room temperature while aslow stream of air was continuously passed through it for 26 hr.(Yellow-orange solid began separating after 2 hr.) The mixture was thenkept in a refrigerator for 16 hr before the solid was collected, washedsuccessively with cold H₂O, EtOH, and Et₂O before it was dried toconstant weight in vacuo over P₂O₅ at 25° C. [The crude product mixture(47 g) was weighed in order to obtain an estimate of the volume of 48%HBr required to form hydrobromide salts.] A mechanically stirred mixtureof the dried solid and EtOH (6.05 l.) was heated to 70° C., and asolution of 48% HBr (28 ml) in EtOH (490 ml) was added in a thin streamwhile the mixture was maintained at 70-75° C. The mixture was thenrefluxed for about 5 min with rapid stirring while nearly all of thesolid dissolved. The hot solution was treated with Norit and filteredthrough a Celite mat. The clear yellow filtrate was kept in arefrigerator overnight while a first crop of orange-colored solidseparated. The collected solid was washed with EtOH, then dried in vacuo(56° C. over P₂O₅) to give 17.2 g of product. The filtrate wasconcentrated by evaporation (rotary evaporator, H₂O aspirator, bath to35° C.) to about 2 l. and then refrigerated to give a second crop, whichwas dried as before, of 10.2 g; total yield 27.4 g (34%). The ¹H NMRspectrum of this material in CF₃CO₂D showed it to contain a barelydetectable amount of methyl substituted 2,4-diaminopteridine.HBr asevidenced by very weak signals at δ2.83 (CH₃) and δ8.85 (pteridine ringH). Strong signals produced by the desired product occur at δ5.28(6-CH₂O) and δ9.08 (C₇—H). The proportion of desired product to themethyl-substituted contaminant was estimated from the ¹H NMR integralsto be 20:1. The ¹H NMR spectrum also revealed retention of a smallamount of EtOH in the product dried as described but not enough tointerfere with the conversion of it to 2.

Example 5 2,4-Diamino-6-(bromomethyl)pteridine (2) hydrobromide saltfrom (5)

Bromine (59.6 g, 0.373 mole) was added dropwise over a 30-min period toa stirred solution of triphenylphosphine (97.7 g, 0.373 mole) inanhydrous 486 ml of dimethylacetamide (DMAC) kept at about 10° C. (icebath) and protected from atmospheric moisture. (Bromine remaining in thefunnel was rinsed with 10 ml of DMAC). A smooth suspension containingfinely divided, crystalline triphenylphosphine dibromide resulted. The2,4-diamino-6-pteridinemethanol.HBr (2) (25.4 g, 0.093 mole) describedabove was added in one portion through a powder funnel (with the aid of10 ml DMAC). The ice bath was removed, and the stirred mixture wasallowed to warm to 20-25° C. After about 1 hr, complete solution hadoccurred. The solution, which gradually developed a dark-red color, waskept at 20-25° C. for 1 hr longer and was then chilled (ice bath) beforeit was treated with EtOH (72 ml). After overnight refrigeration, thesolvents were removed by evaporation in vacuo. The dark, semisolidresidue was stirred with two 300-ml portions of benzene (to removetriphenylphosphine oxide), and each portion was removed from thebenzene-insoluble product by decantation. The solid that remained wasdissolved with stirring in glacial AcOH (660 ml) which had beenpreheated to 80° C. The mixture was kept in a bath at 80° C. untilsolution was complete. A tan crystalline solid separated as the darksolution was allowed to cool. Overnight refrigeration caused the AcOH topartially freeze. When it had thawed, the solid was collected, washedwith chilled AcOH followed by Et₂O, and dried in vacuo (over P₂O₅ andNaOH pellets) at successive temperatures of 25° C., 56° C., and 110° C.(The higher temperature was necessary for complete removal of AcOH). Theyield was 15.3 g (49%). (Some runs afforded 60% yield). This sample wasfurther purified by reprecipitation from MeOH solution (Norit) byaddition of Et₂O followed by drying in vacuo (25° C., P₂O₅), yield 13.0g (42%) of pale-yellow solid. Spectral data: λmax, nm (ε×10⁻³), 0.1 NHCl, 249 (17.3), 339 (10.5), 353 (sh); pH 7, 258 (21.2), 370 (6.87); 0.1N NaOH, 258 (21.5), 370 (6.94); ¹H NMR (CF₃CO₂D), δ 4.70 (s, 2, CH₂) andδ9.08 (s, 1, C₇—H); estimated proportion relative to themethyl-substituted contaminant, 25:1. The preparation of 2 describedabove is typical of several runs that gave similar yields of materialwhose ¹H NMR spectra differed only slightly in the estimated proportionof 2 with respect to the methyl-substituted contaminant. The proportionsusually ranged from 16:1 to 25:1, which corresponds to a percentage of 2of 94 to 96%.

Example 6 2,4-Diamino-6-(bromomethyl)pteridine (2) hydrobromide saltfrom (1)

A suspension of 5 mmol 2,4,5,6-tetraminopyrimidine dibromide in 50 mlmethanol was treated with a solution of 7.5 mmol β-bromopyruvaldoxime in10 ml of methanol at reflux temperature for 2 h. The2,4-diamino-6-(bromomethyl)pteridine was collected after neutralizationwith concentrated NH₃ at room temperature, washed with methanol, etherand dried at 100° C. in an oven. ¹H NMR (250 MHz, ppm, DMSO-d₆), δ 8.84(s, 1H, C₇—H). The yield was 88%.

Example 7 Active Pharmaceutical Ingredient Containing Aminopterin (4)

A mixture of 2 (168 mg, 0.500 mmole) and N-(4-aminobenzoyl)-L-glutamicacid, compound 3 (400 mg, 1.50 mmoles) in DMAC (2 ml) was stirred at 25°C. under N₂ in a stoppered flask protected from light. Solution occurredafter 2 hrs. After 18 hrs, the orange solution was mixed with H₂O (15ml) with stirring to give a finely divided, yellow precipitate. Themixture was centrifuged, and the supernatant removed by decantation. Theyellow solid was stirred with four 15-ml portions of H₂O, each of whichwas removed by decantation after centrifugation. The solid was thensuspended in EtOH (15-20 ml), collected by filtration, washed with Et₂O,and dried in vacuo (25° C., P₂O₅) to give hydrated 4 in 68% yield (160mg). Anal. Calcd for C₁₉H₂₀N₈O₅. 1.75H₂O: C, 48.36; H, 5.02; N, 23.74.Found: C, 48.72; H, 4.91; N, 23.36. Spectral data: λmax, nm (ε×10⁻³),0.1 N HCl, 244 (18.2), 290 (20.5), 335 (11.0); pH 7, 260 (26.7), 283(25.5), 370 (8.00); 0.1 N NaOH, 260 (26.9), 283 (25.3), 370 (8.00); ¹HNMR (DMSO-d₆), δ2.02 (m, 2, CHCH₂CH₂), 2.32 (m, 2, CH₂CO₂H), 4.36 (m, 1,NHCHCO₂H), 4.52 (s, 2, CH₂N), 6.85 (m, 4, 2 phenylene protons plus NH₂),7.72 (m, 2, phenylene), 7.86 (broad s, 2, NH₂), 8.13 (d, 1, NHCO), 8.72(s, 1, C₇—H). Examination by tlc revealed one uv-absorbing spot and nofluorescence at any point. The product may be used directly as theactive pharmaceutical ingredient, but is occasionally subjected to oneor more re-crystallizations from water or formamide to improve theaminopterin purity slightly. The active pharmaceutical ingredient isstored in the presence of desiccant.

Examples 8-13 Analysis of Active Pharmaceutical Ingredient

Three different 1 mM solutions of a first batch of the activepharmaceutical ingredient prepared according to Example 59 were preparedby weighing out 2.3 mg, 4.5 mg and 1.8 mg of the active pharmaceuticalingredient as if it were 100% aminopterin (FW 440.42 g/mole), anddissolving it into 5.223 ml, 10.218 ml, and 4.087 ml of 0.001 N NaOH,respectively. Twenty μl of each 1 mM solution was subjected to HPLCanalysis by injecting each onto a C18 column (Waters μBondapak 125 Å, 10μm, 3.9×150 mm) using an isocratic mobile phase consisting of 5 mM PicA,10 mM NH₄H₂PO₄ and 20% methanol at a pH of 6.8. The flow rate of themobile phase was 1 ml/min, and the analysis was performed at roomtemperature. Using a Waters 996 PDA UV spectrophotometer, absorbancedata from 210 nm to 400 nm was captured. The data was analyzed with theWaters Millennium software by extracting the chromatogram at 282 nm andcalculating the peak area percentages. The data was also analyzed byextracting the spectra of individual peaks, allowing the identificationof pABAGlu, folic acid, aminopterin and pterin species by theircharacteristic spectra.

The average of the HPLC analyses of the three separate 1 mM solutionsrevealed that the first batch of active pharmaceutical ingredientconsisted of approximately 96.27 area % aminopterin and 3.73 area %impurities, wherein the impurities were made up of 0.23 area % folicacid, 2.18 area % N-(4-aminobenzoyl)-L-glutamic acid (i.e. pABAGlu), and0.92 area % of a pterin, probably 2,4-Diamino-6-(bromomethyl)pteridine,and 0.4 area % of at least two other unidentified impurities (Example 8,Table I). A similar HPLC analysis revealed that a second batch of activepharmaceutical ingredient consisted of 97.23 area % aminopterin and2.775 area % impurities, wherein the impurities were made up of 1.82area % folic acid, 0.556 area % N-(4-aminobenzoyl)-L-glutamic acid (i.e.pABAGlu), 0.343 area % pterin, probably2,4-diamino-6-(bromomethyl)pteridine), and 0.056 area % of anunidentified impurity (Example 9, Table I). By comparison, the prior artactive pharmaceutical ingredients contained aminopterin and variableamounts of impurities ranging up to 41% (Examples 10-13, Table I).

Example 14 Pharmaceutical Compositions

This example illustrates the preparation of pharmaceutical compositionssubstantially free of impurities. As used in this example, “API” meansan active pharmaceutical ingredient substantially free of impurities,wherein the antifolate in the API is aminopterin, or a pharmaceuticallyacceptable salt thereof. The aminopterin purity of the API is used toestablished how much API is required in the pharmaceutical compositionto obtained a desired final amount of aminopterin, or a pharmaceuticallyacceptable salt thereof, in the pharmaceutical composition.

Tablets: Surface deposit aminopterin by combining 16.8 grams API, 791.0grams microcrystalline cellulose, 553.7 grams lactose monohydrate, 1.5 gNaOH and 632 grams of sterile water. Mix and dry overnight. Add 1311grams of this surface deposited aminopterin to 171 grams of lactose, 3.9grams colloidal silicon dioxide, 46.2 grams sodium croscarmellose, and7.7 grams magnesium stearate to provide a total weight 1540 grams.Compress into approximately 15,000 tablets using a tableting machine,wherein each tablet weighs approximately 100 mg and contains about 1 mgaminopterin.

Gelatin capsules: Prepare by mixing 0.5 grams of API with 0.5 gramsmagnesium stearate, and 99 grams of lactose. Dispense 100 mg of thismixture into hard-shell gelatin capsules to provide each capsule withabout 0.5 mg of aminopterin.

Suspension: An aqueous suspension is prepared for oral administration sothat each 5 ml contains about 2 mg aminopterin by mixing 40 mg of APIwith 20 grams of sodium carboxymethyl cellulose, 0.5 grams of sodiumbenzoate, 100 grams of sorbitol solution U.S.P., and 2.5 ml of vanillin.

Liquid for injection or oral administration: Clean glassware to be usedwith 2 M NaOH for 3-5 minutes and the rinse thoroughly with deionizedwater. Prepare a first saline buffer by adding 2.7017 grams of dibasicsodium phosphate, USP to 5 liters of 0.9% sodium chloride for injection,USP. Prepare a second saline buffer by adding 1.39 grams of monobasicsodium phosphate, USP to 1 liter of 0.9% sodium chloride for injection,USP. While stirring the first saline buffer solution, slowly add thesecond saline buffer solution until the final pH is 7.9-8.1. Record thefinal volume. Add sufficient API to provide 0.4 mg/ml aminopterin (i.e.2 mg aminopterin per 5 ml) and filter through a 0.2 micron membranefilter and package under sterile conditions in 10-ml vials eachcontaining 5 ml. Use within 2 months.

Injectable: A parenteral formulation is prepared by mixing 0.200 gramsof API with 20.0 grams of propylene glycol, 20.0 grams of polyethyleneglycol 400, and 1.0 gram of polysorbate 80. A sufficient quantity of0.9% saline solution is then added with stirring to provide 100 ml ofsolution which is filtered through a 0.2 micron membrane filter andpackaged under sterile conditions in 2-ml vials each containing 1 ml, ora total of about 2 mg/vial aminopterin. The solution is diluted 10-foldwith 0.9% saline solution prior to administration via an intravenousroute.

Example 15 Mean and Standard Deviation of Dose in PharmaceuticalCompositions

The average dose and the dose uniformity (i.e. standard deviation) ofaminopterin among 10 randomly selected tablets were determined for abatch of 15,000 1 mg aminopterin tablets prepared according to Example14 (Table II). The mean dose and dose uniformity were obtained byscanning spectrophotometry and a radioligand binding assay usingdihydrofolate reductase (DHFR) as binder and methotrexate as a standard.

For scanning spectrophotometry, each of ten tablets are dissolved in11.35 ml of water by rotating in a 15 ml tube for 30 minutes at roomtemperature to make an approximately 200 μM solution (i.e. about 1 mgaminopterin with a FW of 440 g/mole in 11.35 ml). The tubes are spun,and a portion of the supernatant is diluted ten-fold with 0.1 N NaOH toa final concentration of 20 μM. The absorbance of the dilutedsupernatant is read at 282 nm and 260 nm on a Perkin-Elmer Lambda 4Bscanning spectrophotometer (1 cm path length) and the concentration ofthe diluted sample calculated using the relationships, concentration (inμM) at 282 nm=10×(OD₂₈₂/0.264) and concentration (in μM) at 260nm=10×(OD₂₆₀/0.285). The mg/ml at 282 nm and 260 nm is then calculatedusing the relationship, mg/ml=concentration (in μM)×0.00044. The totalmg of aminopterin in each tablet is then determined by multiplying themg/ml determined at 282 nm and 260 nm by 113.5. The mg of aminopterinper tablet is then reported as the average of the mg of aminopterin ineach tablet determined at 282 nm and 260 nm. For example, using theabove procedure, a 1 mg aminopterin tablet yields an OD₂₈₂ of 0.5227,and a calculated concentration (in μM), mg/ml, and total mg ofaminopterin of 19.8 μM, 0.008712 mg/ml, and 0.9888 mg, respectively.Using the above procedure, the same 1 mg aminopterin tablet yields anOD₂₆₀ of 0.5641, and a calculated concentration (in μM), mg/ml, andtotal mg of aminopterin of 19.8 μM, 0.008712 mg/ml, and 0.9888 mg,respectively. The reported amount of aminopterin in the tablet is themean of total aminopterin in the tablet calculated at 282 nm and 260 nm,or 0.9888 mg.

The radioligand binding assay was performed essentially as describedpreviously for methotrexate [see Kamen et al., Anal. Biochem. 70:54,1976 and Ratliff et al., J. Clin. One. 16:1458, 1998]. Briefly, astandard curve was developed for 0.2 to 1.0 pmol methotrexate binding topartially purified chicken liver DHFR, wherein binding of a known amountof non-radioactive methotrexate results in the displacement of an amountof tritiated methotrexate (i.e. radioactive ³H-methotrexate).Aminopterin and methotrexate are equivalent in the assay in terms oftheir displacement and binding, and the absolute detection limit of theassay is 0.05 to 0.10 pmol of antifolate. After the standard curve isestablished, a portion of the 200 μM supernatant prepared above for anaminopterin tablet is further diluted 10,000 using two serial 100-folddilutions with water to provide an approximately 20 nM solution ofaminopterin. The DHFR assay is performed with 100 μL of the 20 nMsolution, as well as several 2-fold serial dilutions, and the resultexpressed as pmol/ml aminopterin. The total number of pmol ofaminopterin is calculated using the relationship, pmol/ml×10,000×11.35.The total number of mg in the tablet is then calculated using therelationship, total pmol×(10⁻⁶ μmole/pmol)×0.44 mg/μmole.

As determined by scanning spectrophotometry, the average dose and thedose uniformity (i.e. standard deviation) of aminopterin among 10randomly selected tablets from a batch of 15,000 1 mg aminopterintablets was 0.974 mg and 2.271%, respectively (Table II). As determinedby radioligand binding assay, the average dose and the dose uniformity(i.e. standard deviation) of aminopterin among the same 10 tablets was1.01 mg and 3.929%, respectively (Table II).

The present invention is not to be limited in scope by the specificembodiments disclosed in the examples which are intended asillustrations of a number of aspects of the invention and anyembodiments which are functionally equivalent are within the scope ofthis invention. Indeed, various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art and are intended to fall within appendedclaims.

A number of references have been cited, the entire disclosures of whichare incorporated herein by reference.

TABLE I AMT total Composition of Example AMT purity impurities totalimpurities  8 96.27% 3.730% FA (0.23%); pABAGlu (2.18%); pterins(0.92%); other (0.4%)  9 97.23% 2.775% FA (1.82%); pABAGlu (0.556%);pterins (0.343%); other (0.056%) 10*^(,a) 70-80% 20-30% not specified11*^(,b)   <80%   >20% FA (20%); others unspecified 12*^(,c)   80%   20%FA (15%); pterins (5%) 13*^(,d)   59%   41% not specified Percentagesare HPLC peak areas. Abbreviations: FA, folic acid; AMT, aminopterin;and pABAGlu, N-(4-aminobenzoyl)-L-glutamic acid. *Not part of thesubject invention. ^(a)Seeger, et al., J. Am. Chem. Soc. 71: 1753, 1949.^(b)Heinrich et al., J. Am. Chem. Soc. 75: 5425. ^(c)Loo, J. Med. Chem.8: 139, 1965. ^(d)Sirotnak and Donsbach, Biochem. Pharmacol. 24: 156,1975.

TABLE II aminopterin dose aminopterin dose by radioligand Random byspectrophotometry binding assay Tablet (mg) (mg) 1 0.99 1.03 2 0.98 0.933 0.99 1.04 4 0.97 1.04 5 0.94 0.96 6 0.96 1.05 7 0.99 0.99 8 0.94 1.039 0.97 1.01 10  1.01 1.03 mean 0.974 1.01 standard 2.271% 3.929%deviation

1. A method for treating an inflammatory disorder in a patient,comprising administering to said patient a therapeutically effectiveamount of aminopterin, or a pharmaceutically acceptable salt thereof, inuninterrupted cycles, wherein the therapeutically effective amount ofaminopterin, or pharmaceutically acceptable salt thereof, administeredper week is between 0.0005 and 0.07 mg per kilogram of patient bodyweight, and wherein the inflammatory disorder is selected from the groupconsisting of rheumatoid arthritis, juvenile rheumatoid arthritis,psoriasis, psoriatic arthritis, atopic dermatitis, inflammatory boweldisease, bronchopulmonary dysplasia, and canine atopic dermatitis.
 2. Amethod of claim 1, wherein the number of uninterrupted cycles is atleast
 24. 3. A method of claim 1, wherein the periodicity of theuninterrupted cycles is weekly.
 4. A method of claim 1, wherein thenumber of doses in each cycle is
 2. 5. A method of claim 1, wherein thenumber of doses in each cycle is
 1. 6. A method of claim 1, comprisingthe additional step of using a second drug in a combination therapy. 7.A method of claim 6, wherein the second drug is folic acid.
 8. A methodof claim 1, wherein the amount of aminopterin given in each cycle isless than 0.07 mg aminopterin per kilogram of patient body weight.
 9. Amethod of claim 1, wherein the therapeutically effective amount ofaminopterin, or pharmaceutically acceptable salt thereof, is between 0.1and 2.0 mg for each cycle.
 10. A method of claim 1, wherein thetherapeutically effective amount of aminopterin, or pharmaceuticallyacceptable salt thereof, is a pharmaceutical composition, and whereinthe pharmaceutical composition is a tablet.
 11. A method for treating aninflammatory disorder in a patient, comprising administering to saidpatient a therapeutically effective amount of aminopterin, or apharmaceutically acceptable salt thereof, wherein the therapeuticallyeffective amount of aminopterin, or pharmaceutically acceptable saltthereof, administered per week is between 0.0005 and 0.07 mg perkilogram of patient body weight, and wherein the inflammatory disorderis selected from the group consisting of rheumatoid arthritis, juvenilerheumatoid arthritis, psoriasis, psoriatic arthritis, arthritis, atopicdermatitis, inflammatory bowel disease, bronchopulmonary dysplasia, andcanine atopic dermatitis.
 12. A method of claim 11, wherein the amountof aminopterin, or pharmaceutically acceptable salt thereof,administered is between 0.0005 and 0.05 mg per kilogram of patient bodyweight.
 13. A method of claim 11, wherein the amount of aminopterin, orpharmaceutically acceptable salt thereof, administered per week isbetween 0.001 and 0.07 ing per kilogram of patient body weight.
 14. Amethod of claim 11, wherein the amount of aminopterin, orpharmaceutically acceptable salt thereof, administered per week isbetween 0.005 and 0.07 mg per kilogram of patient body weight.
 15. Amethod of cLaim 11, comprising the additional step of using a seconddrug in a combination therapy.
 16. A method of claim 15, wherein thesecond drug is folic acid.
 17. A method of claim 11, wherein thetherapeutically effective amount of aminopterin, or pharmaceuticallyacceptable salt thereof, is a pharmaceutical composition, and whereinthe pharmaceutical composition is a tablet.